Studies On Preparation And Preliminary Application Of Recombinant Lactobacillus Plantarum Expressing Porcine Interleukin-10

Animal husbandry is an important part of modern agriculture industry and is also strategic adjustment competitive industries of our country agriculture and rural economic structure.Our country is the big country of pig production and pork consumers. The mode of intensive cultivation resolve the consumption of the pork products against high population density, but the highly intensive farming conditions induce the dropping of pig immunity and anti-stress, increasing the emissions of pathogenic microorganisms and the opportunity of contacting pathogenic microorganisms, which increase the mortality rates and downregulate the growth speed, breeding income and the supply of pork products etc. Thus, improving the immune level of pigs and increasing the ability of anti-pathogenic microorganisms provide the important practical significance for promoting the continuous and healthy development of pig industry, the level of rural economic growth and guaranteing the high quality pork supply.Interleukin-10(interleukin-10, IL-10), as a unique cytokines, is firstly found in the secretion of Th2cells in mice.It cause the attention of scholars both at home and abroad because it can suppresse synthesis of cytokines producing Thl cells.Study found that IL-10is produced by activated immune cells, especially mononuclear macrophages and subpopulation of T cells, consisting of Tr1,Treg and Thl cells. IL-10is also produced by B cells, dendritic cells, keratinocytes and mast cells [1]. IL-10can restrain the production and release of inflammation factors IL-2and IFN-y from Thl cells,which has a strong anti-inflammatory and immune suppression activity and also can reduce the expression of MHCⅡ. In addition, IL-10can stimulate the proliferation, differentiation and producing antibodies of B cells. IL-10, as a immunoregulation factor of the rare two-way of the body, its unique immunoregulation role has been hot spot. This study we clone IL-10gene from pig to E. coli-lactic acid bacteria expression vector, and then electrotransformed into the lactic acid bacteria.Therefove,we obtain recombinant lactic acid bacteria with the activity of immune regulation,which provide certain significance for the development of lactic acid bacteria preparations to protect animal safety production and animal food safety production. Specific research contents and the results are as follows:The total RNA were extracted from pig spleen, reverse transcriptase into cDNA, according to the pig source interleukin10(pIL-10) gene sequences form the publication of GenBank, designed and synthesized primer combining with the shuttle express vectors site inserted by purpose gene into.We successfully expand pIL-10in vitro by RT-PCR. Connected pIL-10genes with pMD18-T carrier and transited into the E.coli DH5a, constructing the cloning vector pMD18-T-pIL-10, extraction plasmid.We identificated the sequence through the PCR, restriction enzymes and sequencing method, and gene sequences homology are the same with sequences form the publication of GenBank. Therefore,We successfully constructs pMD18-T-pIL-10cloning vector.The recombinant plasmid pMD18-T-pIL-10was digested with restriction enzyme generating IL-10gene, and subcloned into the vector pSIP409.The recombinant plasmid pSIP409-pIL-10was transferred into the Lb.plantarum NC8by electroporation. The recombinant lactic acid bacteria was induced by Sakacin-P(SppIP) and identified by SDS-PAGE analysis. The recombinant lactic acid bacteria express18kDa protein and the results are consistent with the expectations. The Western blot analysis shows that IL-10possess immunogenicity.Weaning piglets were inoculated with Lactic acid bacteria continuously for three weeks, in the second week, and were inoculated with ETEC O149:K88for one week until the piglets fall ill. The results show that, comparing with the control group, the recombinant lactic acid bacteria pSIP409-IL-10can enhance the cell immune level, stimulating the production of antibody and inducing local mucosa reaction, downregulation the level of inflammatory factor TNF-a and at the same time promoting the expression of anti-inflammatory factor IL-4.It can obviously improve the changes of piglets histopathological. The recombinant strains were successfully field planting in the intestinal through the antigen positioning and detection of intestinal content to IL-10and TNF-α.